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Archives of Razi Institute, Vol. 73, No. 1 (2018) 27-38 Copyright © 2018 by
Razi Vaccine & Serum Research Institute
Original Article
High-level expression of tetanus toxin fragment C in Escherichia coli
Aghayipour∗∗∗∗, K., Teymourpour, R.
Department of Genomics and Genetic Engineering, Razi Vaccine and Serum Research Institute, Agricultural Research,
Education, and Extension Organization, Karaj, Iran
Received 15 June 2016; accepted 18 February 2017
Corresponding Author: Khosrow@rvsri.ac.ir
ABSTRACT
Fragment C is the C-terminal domain of the heavy chain of tetanus toxin that can promote the immune response
against the lethal dose of this toxin. Therefore, this portion can be considered as a candidate vaccine against
tetanus infection, which occurs by Clostridium tetani. The present study aimed to compare the expression of
tetanus toxin fragment C in Escherichia coli BL21 (DE3) pLysS cells having a high tolerance to toxins between
two different expression vectors, namely pET22b and pET28a, using the sodium dodecyl sulfate polyacrylamide
gel electrophoresis and western blot analyses. After DNA extraction from Harvard CN49205 strain of C. tetani,
the gene of interest was amplified using polymerase chain reaction, and then sequenced and cloned into the
expression vectors of pET22b and pET28a, transformed into competent BL21 (DE3) pLysS cells, and finally
expressed using an optimized protocol. The cells were induced with isopropyl β-D-1-thiogalactopyranoside
(IPTG) at four different incubation temperatures (i.e., 37, 33, 30, and 25 °C) and three different incubation times
(i.e., 1, 2, and 3 h). Although the SDS-PAGE and western blot analyses confirmed the expression of the
recombinant fragment C (r-fragment C) ligated into both of the expression vectors, pET28a showed a higher r-
fragment C expression level than the other vector (38.66 mg/L versus 32.33 mg/L, P<0.05). An optimal
expression condition was acquired 3 h after 1 mM IPTG induction at 25 °C. The results demonstrated that E.
coli BL21 (DE3) pLysS as an expression host in combination with pET-28a as an expression vector was a more
compatible expression system to express the fragment C of tetanus toxin, compared to E. coli BL21 (DE3)
pLysS/pET-22b expression system. Overall, these results may represent an opportunity to improve the
expression system for the production of tetanus toxin vaccine using recombinant protein strategy.
Keywords: Clostridium tetani, Fragment C, pET22b expression vector, pET28a expression vector, E. coli BL21
(DE3) pLysS
La forte expression du fragment C de la toxine tétanique dans Escherichia coli
Résumé: Le fragment C'est le domaine C-terminal de la chaîne lourde de la toxine tétanique. Ce dernier favorisé
la réponse immunitaire contre la dose létale de cette toxine et peut donc être considéré comme un candidat
potentiel pour le vaccin contre l'infection tétanique causée par Clostridium tetani. Dans cette étude, l'expression
du fragment C de la toxine tétanique dans des cellules BL21 (DE3) pLysS d'E. coli ayant une tolérance élevée
aux toxines a été comparée en utilisant deux vecteurs d'expression différents, pET22b et pET28a, par SDS-
PAGE et western blot. Après l’extraction de l'ADN de la souche Harvard CN49205 de C. tetani, le gène d'intérêt
a été amplifié par PCR, séquencé, cloné dans les vecteurs d'expression, pET22b et pET28a, transformé dans des
cellules compétentes BL21 (DE3) pLysS et enfin exprimé selon un protocole optimisé. Les cellules ont été
induites avec IPTG à quatre températures d'incubation différentes (37, 33, 30 et 25° C) et trois temps
d'incubation différents (1 à 3 h). Bien que l'expression du fragment recombinant C (fragment C) ligaturé dans les
CORE Metadata, citation and similar papers at core.ac.uk
Aghayipour & Teymourpour / Archives of Razi Institute, Vol. 73, No. 1 (2018) 27-38 28
INTRODUCTION
Tetanus is an infection caused by Clostridium tetani,
which is an obligate anaerobic spore-forming bacterium
(Gil et al., 2001). Tetanus toxin is a potent neurotoxin
that is intracellularly synthesized by C. tetani as a
single 150 kDa polypeptide chain. Following cell lysis,
the toxin is released into the medium and cleaved by
endogenous proteases generating a 50 kDa N-terminal
light chain (fragment A) disulfide bonded to a 100 kDa
C-terminal heavy chain (fragments B and C)
(Bruggemann and Gottschalk, 2004). Fragment C, the
50 kDa C-terminal portion of the heavy chain, has
ganglioside (Helting and Zwisler, 1977) and protein
(Schiavo et al., 1991) binding activities in the tissues of
neural origin. These gangliosides are considered to be
potential relevant eukaryotic cell receptors (Herreros et
al., 2000). Fragment C is completely nontoxic in
animals, whereas fragment B retains some residual
toxicity at high doses (Helting et al., 1977). Fragment C
has been demonstrated to retain the ganglioside binding
activity of intact tetanus toxin (Morris et al., 1980).
Therefore, purified fragment C is used to successfully
immunize the animals against tetanus. This indicates
that the entire molecule is not essential for protection
(Helting and Nau, 1984). It seems that this fragment
can be used as a novel efficient vaccine without any
neurotoxicity. In some studies, the recombinant
fragment C (r-fragment C) was expressed alone or in
fusion with other antigens on different strains of
Escherichia coli (Fairweather and Lyness, 1986;
Makoff et al., 1989; Halpern et al., 1990; Ribas et al.,
2000; Yousefi et al., 2013), Salmonella (Chatfield et
al., 1992), Lactobacillus (Maassen et al., 1999),
Bordetella (Stevenson and Roberts, 2004), yeast cells
(Romanos et al., 1991), and cultured insect cells
(Charles et al., 1991), which in some cases resulted in
the induction of protective antibodies against tetanus
toxin. However, each of these studies encountered
some deficiencies, such as the presence of several
fortuitous polyadenylation sites within r-fragment C in
S. cerevisiae (Romanos et al., 1991), presence of
glycosylation sites in S. cerevisiae and Pichia pastoris,
which would result in inactive antigen (Romanos et al.,
1991), phenotypic alterations, including chlorotic
phenotype and male sterility in tobacco leaves
(Tregoning et al., 2003), low ganglioside binding
activity of r-fragment C of baculovirus expression
system (Charles et al., 1991), and ethical issues
regarding live vector vaccines. E. coli expression
system also has some problems, such as low expression
level and low solubility rates of r-fragment C. Despite
the many attempts made to improve the expression
level and solubility rate of r-fragment C (Fairweather et
al., 1986; Makoff et al., 1989; Halpern et al., 1990;
Ribas et al., 2000; Wang et al., 2008; Yousefi et al.,
2013), the suggested solutions in this area have not
been analyzed yet. It seems that some limitations in the
deux vecteurs d'expression ait été confirmée par les analyses SDS-PAGE et western blot, pET28a a montré une
expression plus élevée du fragment r C comparé à pET22b (38,66 mg / l contre 32,33 mg / l, p <0,5). La
condition optimale d'expression a été obtenueà 5° C, IPTG 1 mM, et 3 h après l’induction de l’IPTG. Ces
résultats ont démontré que l’hôte d'expression E. coli BL21 (DE3) pLysS en combinaison avec le vecteur deux
vecteurs d'expression ait été confirmée par les analyses SDS-PAGE et western blot, pET28a a montré une
expression plus élevée du fragment r C comparé à pET22b (38,66 mg / l contre 32,33 mg / l, p <0,5). La
condition optimale d'expression a été obtenueà 5° C, IPTG 1 mM, et 3 h après l’induction de l’IPTG. Ces
résultats ont démontré que l’hôte d'expression E. coli BL21(DE3) pLysS en combinaison avec le vecteur
d'expression pET-28a représentait le système d'expression le plus compatible pour exprimer le fragment C de la
toxine tétanique. Dans l’ensemble, ces résultats montrent qu’il est possible d’améliorer la production de vaccin
contre la toxine tétanique à base de protéines recombinantes en optimisant leur système d'expression.
Mots-clés: Clostridium tetani, Fragment C, vecteur d'expression pET22b, vecteur d'expression de pET28a, E.
coli BL21 (DE3) pLysS
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29
expression level of r-fragment C are related to the
probable toxicity of this antigen to E. coli as the
expression host. On the other hand, the low solubility
rate of this protein is probably due to its high tendency
to form inclusion bodies in overproduction condition
(Miyake et al., 2007). The two major factors that
reduce the production rate of the recombinant proteins,
such as r-fragment C, are the formation of inclusion
bodies resulting in a low solubility rate (Miyake et al.,
2007) and degradation by lon and ompT outer
membrane proteases (Goff and Goldberg; Grodberg
and Dunn, 1988). Another problem in this area is the
limitations related to the traditional expression method
(Sambrook and Russell, 2001). Therefore, it could be
highly desirable to investigate the expression of r-
fragment C in the BL21 (DE3) pLysS strain of E. coli
containing a pLysS plasmid with a high tolerance to
toxins and lacking lon and ompT outer membrane
proteases and an expression vector with a strong
promoter using a modified protocol. With this
background in mind, the present study, aimed to
investigate the expression of the gene encoding
fragment C of tetanus toxin in E. coli BL21 (DE3)
pLysS using pET22b and pET28a vectors. In addition,
a comparative study was performed between pET22b
and pET28a expression vectors in order to determine
the plasmid that is more compatible with E.coli BL21
(DE3) pLysS cells for expressing r-fragment C. We
also used a recently-optimized expression method in
our laboratory (Bahreini et al., 2014) for the
overexpression of r-fragment C. Moreover, different
temperature and time levels were analyzed in order to
determine an optimal expression condition, which is an
important factor in the overexpression and
overproduction of a recombinant protein.
MATERIALS AND METHODS
Polymerase chain reaction, cloning, and DNA
sequencing. Total DNA was extracted from Harvard
CN49205 strain of C. tetani using the High Pure PCR
Template Preparation Kit (Roche Diagnostic,
Germany). The gene encoding fragment C of tetanus
toxin was amplified using specific primers, including
5'GGAATTCCATATGAAAAATCTGGATTGTTGG
GT3' as forward primer containing NdeI restriction site
(shown as underlined letters) as well as
5'CCGCTCGAGTTAATCATTTGTCCATCCTT3' and
5'CCCAAGCTTTTAATCATTTGTCCATCCTT3' as
reverse primers, including XhoI and HindIII restriction
sites, respectively (shown as underlined letters), and
stop codon (shown as italic and bold letters). Based on
the kind of the reverse primer (XhoI or HindIII), two
different PCRs with similar conditions were performed.
The PCRs were carried out with 300 ng of total DNA
in a final volume of 50 µl containing 10X PCR buffer,
0.2 mM dNTP (Roche Diagnostics, Germany), 1
unit/reaction of Pfu DNA polymerase (Fermentas,
Russia) combined with 0.5 unit/reaction of Taq DNA
polymerase (Fermentas, Russia), 1.5 mM MgCl2, 10
pmol/reaction of each forward and reverse primers, and
double-distilled H2O up to the total volume of 50 µl.
Both of the PCR reactions were performed by a Gene
Cycler (Bio-Rad, USA) with the following thermo-
cycling profile: initial denaturation at 95 °C for 5 min,
followed by 5 cycles of 95 °C for 1 min, 50 °C for 1
min, and 72 °C for 1 min, and then 30 cycles of 95 °C
for 1 min, 57 °C for 1 min, and 72 °C for 1 min. The
final extension was 10 min at 72 °C. The PCR products
were visualized by electrophoresis on 1% agarose gel.
Subsequently, they were purified from agarose gel by
the High Pure PCR Template Preparation Kit and
ligated into pTZ57R/T cloning vector (Fermentas,
Germany). The ligated mixtures were transformed into
E. coli DH5α (Invitrogen, USA) competent cells using
the heat shock method (Sambrook and Russell, 2001).
Then, the recombinant transformed colonies were
screened on lysogeny broth (LB) agar media containing
100 µg/mL ampicillin, 20 mg/mL5-bromo-4-chloro-3-
indolyl-β-D-galactopyranoside, and 0.1 M IPTG. Two
different recombinant pTZtetc plasmids were extracted
using the High Pure Plasmid Isolation Kit (Roche
Diagnostic, Germany). Then, the sequencing was
Aghayipour & Teymourpour / Archives of Razi Institute, Vol. 73, No. 1 (2018) 27-38 30
performed in both directions using T7 promoter and
M13 (-20) primers, a BigDye Terminator Cycle
Sequencing Reaction Kit (Applied Biosystems,
Canada), and an ABI 3730 XL DNA Analyzer
(BioNeer, South Korea). The obtained nucleotide
sequence of the gene encoding fragment C was
analyzed using BioEdit software (Hall, 1999).
Subcloning into expression vectors and gene
expression. One of the two distinctive recombinant
pTZtetc plasmids harboring NdeI and XhoI restriction
sites was ligated into pET22b expression vector
(Novagen, USA) at NdeI and XhoI cloning sites. The
other recombinant pTZtetc plasmid containing NdeI
and HindIII restriction sites was ligated into pET28a
expression vector (Novagen, USA) at NdeI and HindIII
cloning sites. In order to express r-fragment C, initially,
the recombinant pET22b and pET28a expression
vectors were separately transformed into E. coli BL21
(DE3) pLysS competent cells using the heat shock
method. Subsequently, the mixtures were cultured on
(LB) agar plates containing 50 µg/mL ampicillin and
34 µg/mL chloramphenicol obtained from recombinant
E. coli BL21 (DE3) pLysS transformed with pET22b,
as well as 30 µg/mL kanamycin and 34 µg/mL
chloramphenicol obtained from recombinant E. coli
BL21 (DE3) pLysS transformed with pET28a. In order
to overexpress the gene of interest, we used an
optimized expression method reported in our previous
study (Bahreini et al., 2014) rather than the traditional
method described by Sambrook and Russell (2001). To
this end, three steps were considered for the bacterial
growth at 37oC in a shaking incubator at 200 rpm
(Bahreini et al., 2014). At the first step, a single colony
from each of the two recombinant transformed E. coli
BL21 (DE3) pLysS cells, cultured on the LB plates,
was inoculated in 5 mL of LB broth supplemented with
1% w/v glucose (50 µg/mL ampicillin and 30 µg/mL
kanamycin obtained from pET22b and pET28a,
respectively). The resulting mixtures were incubated
for 7 h. At the second step, the first-step media were
transferred into 100 mL of LB broth (1% w/v glucose),
containing 300 µg/mL ampicillin and 200 µg/mL
kanamycin obtained from pET22b and pET28a,
respectively, and incubated overnight. At the third step,
the overnight media were exchanged by centrifuging
(1200 g, 4 °C for 5 min). Subsequently, the fresh LB
media (1% w/v glucose), containing 50 µg/mL
ampicillin and 30 µg/mL kanamycin obtained from
pET22b and pET28a, respectively, were added to the
bacterial pellets and diluted until obtaining an optical
density(OD) of 10 at 600 nm. The last media were
incubated for 75 min in order to adapt bacteria to the
fresh media until adding inducer and beginning protein
expression. Afterwards, each medium was incubated
with 1 mM IPTG at four different incubation
temperatures (i.e., 37, 33, 30, and 25 °C) and three
different incubation times (i.e., 1 to 3 h after IPTG
induction) in order to determine the optimal condition
for the expression of r-fragment C.
Immunoblotting. Prior to IPTG induction and 1, 2,
and 3 h after that, sampling from each medium was
performed, and the cells from each sample were
immediately harvested by centrifuging at 10,000 g and
4 °C for 5 min. Each of the harvested bacterial cell
pellets was diluted and lysed in 5X sample buffer
(Laemmli, 1970). The total protein concentration in cell
lysate was determined by the Bradford Protein Assay
Reagent Kit (Bio-Rad, USA). For the SDS-PAGE
analysis, the protein samples were loaded on a 10×10
cm and 12.5% SDS-PAGE gel following the method
described by Laemmli (1970). The mixtures were
electrophoresed using Protean II xi cells (Bio-Rad,
USA) in electrophoresis buffer (Laemmli, 1970) for 24
h at 30 V. The gel was stained with coomassie brilliant
blue R-250. Finally, the specific band of r-fragment C
protein was cut out from the gel, and its concentration
was evaluated using Bradford assay for both of the
expression vectors. For western blot, fractioned
proteins were transferred into polyvinylidene
fluoride (PVDF) membranes (Roche Diagnostic,
Germany) using an Electro Blot System (Bio-Rad,
USA) in a transferring buffer (pH=8.3), containing 25
mM Tris, 192 mM glycine, and 15% methanol. The
solution was kept overnight at 20 V and 4°C, blocked
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31
with 5% bovine serum albumin (BSA) with gentle
rocking at room temperature for 1.5 h, and washed for
three times in TBST (pH=7.2), containing 20 mM Tris-
HCl, 150 mM CaCl2, and 0.05% W/V Tween 20.
Subsequently, the membrane was probed with horse
anti-tetanus toxin polyclonal antibody (Razi Vaccine
and Serum Research Institute, Karaj, Iran) (1:1000
dilution in TBST) using gentle rocking at ambient
temperature for 1.5 h. The membrane was then washed
four times with TBST and incubated with horse radish
peroxidase-conjugated Anti-Goat IgG (Abcam, UK)
(1:1000 dilution in TBST) using gentle rocking at room
temperature for 1.5 h. After washing the membrane
again, the color was developed using 3'-
Diaminobenzidine.
Statistical analysis. The concentrations of total cell
protein and r-fragment C were determined through the
estimation of the mean and standard deviation.
Additionally, the percent yield was calculated. The t-
test was used to analyze the differences between the
two expression systems in terms of protein
concentration. All data analysis was performed in SPSS
software. P-value less than 0.05 was considered
statistically significant.
RESULTS
Polymerase chain reaction, gene cloning, and
DNA sequence analysis. Two different PCR products,
both of which encoded the fragment C of tetanus toxin,
were obtained using total DNA from C. tetani as a
template and designed primers. The agarose gel
electrophoresis of the two amplicons confirmed them to
be ~ 1.4 kb in size (Figure 1). Both of the PCR
products were then cloned into pTZ57R/T cloning
vector. The recombinant constructs were verified by
means of DNA sequencing and digestion using NdeI
and XhoI restriction endonucleases producing one of
the two amplicons (Figure 2a) and NdeI and HindIII
restriction endonucleases generating the other one
(Figure 2b). DNA sequence analysis confirmed both
amplicons to be exactly 1356 bp in size as reported by
the sequences of C. tetani strain CN3911, a derivative
of Harvard strain (Fairweather and Lyness, 1986) and
Massachusetts strain of C. tetani (Eisell et al., 1986).
Figure 2b. Electrophoresis on
1% (w/v) agarose gel for the
restriction enzyme digestion of
the recombinant cloning vector
(pTZ57R) using NdeI/XhoI,
confirming the insertion of the
gene encoding tetanus toxin
fragment C into this vector.
Lane 1: DNA size marker 1kb
(Fermentase, Germany); lane
2: single digestion product of
recombinant pTZ57R using
NdeI endonuclease; lane 3:
double digestion products of
recombinant pTZ57R using
NdeI and XhoI.
Figure 1. Electrophoresis on
1% (w/v) agarose gel for two
different polymerase chain
reaction products, both
encoding fragment C of
tetanus toxin, verifying the
amplification of the gene of
interest. Lane 1: DNA size
marker 1 kb (Fermentase,
Germany). Lanes 2 and 3:
polymerase chain reaction
products of fragment C gene
obtained by specific primers,
including NdeI/XhoI and
NdeI/HindIII restriction
sites, respectively.
Figure 2a. Electrophoresis on
1% (w/v) agarose gel for two
different polymerase chain
reaction products, both
encoding fragment C of
tetanus toxin, verifying the
amplification of the gene of
interest. Lane 1: DNA size
marker 1 kb (Fermentase,
Germany). Lanes 2 and 3:
polymerase chain reaction
products of fragment C gene
obtained by specific primers,
including NdeI/XhoI and
NdeI/HindIII restriction
sites, respectively.
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Based on the sequence analysis, in the two samples
sequenced in the present study, no mutations or
polymorphisms were found within the gene encoding
fragment C obtained from Harvard CN49205 strain of
C. tetani (DTP department, Razi Vaccine and Serum
Research Institute, Karaj, Iran). The nucleotide
sequence of tetanus toxin fragment C analyzed in the
present study was deposited into the NCBI GenBank
(GenBank ID: FJ917402).
Construction of recombinant expression plasmids.
Figure 3 displays a schematic representation of two
recombinant pET22b and pET28a expression vectors
designed by SnapGene software, version 2.8
(www.snapgene.com). These two vectors both
containing fragment C gene of C. tetani (Figure 3) were
constructed in order to express the r-fragment C protein
in E. coli BL21 (DE3) pLysS. The electrophoresis of
the two recombinant expression vectors on agarose gel
confirmed them to have a higher length than the non-
recombinant ones (~ 6.7 kb versus ~ 5.4 kb For the
recombinant and non-recombinant pET22b, respectively,
and ~ 6.6 kb versus ~ 5.3 kb in the recombinant and non-
recombinant pET28a, respectively) (Figures 2c and 2d).
Expression of r-fragment C. As shown in Figure
4a, the r-fragment C was successfully expressed in E.
coli BL21 (DE3) pLysS as the fusion and native
proteins. The production of the recombinant protein
was detectable after 1 h of IPTG induction and reached
a maximal level after 3 h in both expression vectors.
Furthermore, the high expression level of r-fragment C
was achieved at the incubation temperature of 25 °C
(Figure 4a). The expressed r-fragment C was either
approximately 51.6 kDa as a native protein in pET22b
vector or almost 54 kDa as a fusion protein in pET28a
(Figure 4a). As shown in Table 1, no significant
differences were observed between the two expression
systems in terms of total soluble protein in cell lysate.
However, Bradford analysis showed that the
concentration of r-fragment C obtained by pET28a was
higher than that obtain from pET22b (38.66 mg/L
versus 32.33 mg/L, P<0.5) (Table 1).
Table 1. Mean of total soluble protein and recombinant fragment C
concentrations in cell lysates and percentage of the yield obtained
from the two expression vectors of pET28a and pET22b
Protein source Expression vector
pET28a pET22b P-value
Total soluble protein
(mg/L)
381.67±14.0 395.0±9.85 NS
Recombinant-
fragment C (mg/L)
32.33±3.05 38.66±2.08 P<0.5
Yield (%) 8.5±0.5 9.8±0.4 P<0.5
NS: not-significant, Note: The data presented in this table is related
to the Escherichia coli cultures with log phase of “10” and
expression condition of 25 °C, 1 mM IPTG, and 3 h IPTG
induction.
Western blot analysis. To detect the r-fragment C, a
western blot assay was performed using anti-tetanus
Figure 2c. Electrophoresis on
1% (w/v) agarose gel for the
restriction enzyme digestion of
the recombinant and non-
recombinant expression vector
(pET22b) using NdeI/XhoI,
confirming the insertion of the
gene encoding tetanus toxin
fragment C into this vector. Lane
1: DNA size marker 1kb
(Fermentase, Germany); lane 2:
double digestion product of non-
recombinant pET22b using NdeI
and XhoI; lane 3: double
digestion products of
recombinant pET22b using
NdeI and XhoI.
Figure 2d. Electrophoresis on
1% (w/v) agarose gel for the
restriction enzyme digestion of
the recombinant and non-
recombinant expression vector
(pET28a) using NdeI/HindIII,
confirming the insertion of the
gene encoding tetanus toxin
fragment C into this vector.
Lane 1: DNA size marker 1kb
(Fermentase, Germany); lane
2: double digestion products
of recombinant pET28a using
NdeI and HindIII; lane 3:
double digestion product of
non-recombinant pET28a
using NdeI and HindIII.
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33
toxin-specific polyclonal antibody under reducing
conditions.
Figure 3a. Schematic representation of the recombinant pET22b
expression vector designed by SnapGene software version 2.8. The
gene encoding fragment C of tetanus toxin was cloned into the
NdeI/XhoI restriction sites of pET22b vector.
Figure 3b. Schematic representation of the recombinant pET28a
expression vector designed by SnapGene software version 2.8. The
gene encoding fragment C of tetanus toxin was cloned into the
NdeI/HindIII restriction sites of pET28a vector, in frame with the 6-
histidin tag and thrombin cleavage site in N-terminal proportion of
pET28a.
As shown in Figure 4b, the western blot by
polyclonal horse antisera against r-fragment C of
tetanus toxin resulted in the emergence of only one
band on the membrane in both recombinant native
(51.6 kDa) and fusion (54 kDa) proteins, representing
that horse antibodies specifically bound to the r-
fragment C produced by both expression vectors,
namely pET22b and pET28a.
Figure 4a. Analysis of total cell lysates of transformed E. coli
BL21 (DE3) pLysS, containing recombinant pET22b or pET28a
expression vectors by sodium dodecyl sulfate polyacrylamide (SDS-
PAGE) gel electrophoresis, confirming high expression level of
recombinant fragment C of tetanus toxin. Samples of cell lysates
including total protein content were collected at 24 h intervals after
methanol induction, separated by 12.5% SDS-PAGE gel, and
visualized by staining with coomassie blue R-250. Lane 1: negative
control; lanes 2 and 7: total cell protein obtained from E. coli BL21
(DE3) pLysS cells bearing the recombinant expression constructs in
pET22b and pET28a, respectively, grown in the absence of
isopropyl β-D-1-thiogalactopyranoside (IPTG); lane 3: protein
molecular weight standards (Fermentas, Lithuania); lanes 4, 5, and
6: the same as lane 2, but the samples were taken 1, 2, and 3 h after
IPTG induction, respectively; lanes 8, 9, and 10: the same as lane 7, but the samples were taken 1, 2, and 3 h after IPTG induction,
respectively.
Figure 4b. Analysis of total cell lysates of transformed E. coli
BL21 (DE3) pLysS, containing recombinant pET22b or pET28a
expression vectors by western blot, confirming high purification of
recombinant fragment C of tetanus toxin. Fractioned proteins were
transferred from the SDS-PAGE gel onto a polyvinylidene
difluoride (PVDF) membrane, and a single band representing
recombinant fragment C was detected in both recombinant native
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(51.6 kDa) and fusion (54 kDa) forms; lane 1: schematic
representation of a broad-range protein size marker (Fermentas,
Lithuania); lanes 2 and 3: PVDF membranes probed with total cell
protein from E. coli BL21 (DE3) pLysS cells containing the
recombinant pET28a and pET22b, respectively; lane 4: negative
control.
DISCUSSION
There are many reports confirming the antigenicity of
fragment C of tetanus toxin, since it has been identified
as an immunodominant (Francis et al., 2004; Carlton et
al., 2008) and nontoxic (Helting and Zwisler, 1977;
Helting and Nau, 1984) antigen. Therefore, some
research teams have used this fragment to produce a
recombinant vaccine against tetanus (Maassen et al.,
1999; Ribas et al., 2000; Stevenson and Roberts, 2004).
In the present study, we successfully expressed the r-
fragment C of tetanus toxin in E. coli regarding the
production of recombinant subunit vaccine against
tetanus toxin. Although several studies have hitherto
succeeded to clone and express the fragment C of
tetanus toxin, there are still some problems in the
industrial production of this antigen. A problem
occurring in the overproduction process of many
proteins by a recombinant protein expression system is
the limitations of the traditional, widely used standard
system for protein expression in the shaking flask
cultures described by Sambrook and Russell (2001). In
this regard, some solutions to this problem are
available, which were used in the present study. Based
on the traditional expression methods, the recombinant
protein synthesis is induced, when the (OD600) reaches
to 0.6, at a point about the middle of the exponential
growth. Such low cell densities produce a low yield
value. As a result, a large culture volume is required for
high protein production. On the other hand, the
expression of recombinant plasmid gene is a
biochemical pathway, decreasing the specific rate of
population growth and cell density owing to two
factors. Firstly, this pathway in bacteria is a long
process; therefore, nutrient depletion, accumulation of
waste and toxic metabolites, and inhibitory compounds
in the medium will provide antigrowth conditions, such
as plasmid loss (Hannig and Makrides, 1998), limited
availability of dissolved oxygen (DO), and increased
CO2 levels, which cause significant reduction of
medium pH (Khushoo et al., 2004). Therefore, under
such circumstances, high cell densities cannot be
reached. Secondly, for the high processivity of T7 RNA
polymerase and IPTG induction, most of the
transcription and translation machinery of the cell
might be employed for recombinant protein expression,
resulting in the reduction of bacterial growth rate and
cell density (Sambrook and Russell, 2001). The low
cell density often causes a low protein production. To
overcome these problems, in the present study, we used
a recently-optimized protocol (Bahreini et al., 2014),
which was set in our laboratory using ASNase II as a
recombinant protein model rather than the traditional
protocol (Sambrook and Russell, 2001). This modified
method resulted in a higher number of plasmid copies
in each cell, and subsequently a higher copy number of
the fragment C gene per cell using a high antibiotic
concentration (i.e., 200 and 300 µg/mL) before the
induction time, which would lead to a significant
increase in protein expression per cell (Bahreini et al.,
2014). Furthermore, a fresh medium with a very higher
initial cell density (OD600=10 versus OD600=0.6) at
induction time was provided using a three step-culture,
the third step of which included exchanging the
overnight medium by centrifuging, adding fresh LB
medium to the bacterial pellet, and diluting until
reaching a high cell density. As fragment C is part of a
toxic protein, we supposed that its basal levels may
prevent the full establishment of the plasmid carrying
its codding gene in E. coli strains, which will result in
the incomplete expression of this gene in this
bacterium. On the other hand, one of the major factors
reducing the overproduction of recombinant proteins,
such as r-fragment C, is protein degradation by lon and
ompT outer membrane proteases (Goff and Goldberg;
Grodberg and Dunn, 1988). One way of providing
additional stability to the target genes is to express
them in the host strains containing a compatible
chloramphenicol-resistant plasmid that provides a small
amount of T7 lysozyme, a natural inhibitor of T7 RNA
Aghayipour & Teymourpour / Archives of Razi Institute, Vol. 72, No. 4 (2017) 27-38
35
polymerase (Studier, 1991). In the present study, BL21
(DE3) pLysS strain of E. coli, a high-stringency
expression host, which has the pLysS plasmid, was
used. This plasmid confers resistance to
chloramphenicol. The lysozyme produced by this
plasmid does not interfere with the transformation of
the cells containing it and has no negative effect on the
growth rate. The presence of pLysS increases the
tolerance of expression host in the plasmids with toxic
inserts, and also has the further advantage of facilitating
the preparation of the cell extracts. This strain is also
deficient in the lon protease and lacks the ompT outer
membrane protease. Therefore, fragment C should be
more stable in BL21 (DE3) pLysS than in the host
strains containing the mentioned proteases. An
alternative approach to increase the stability of the
target gene is to use a vector that contains a T7lac
promoter ((Dubendorf and Studier, 1991; Studier,
1991). In this regard, in the present study, two different
expression vectors, namely pET-28a and pET-22b,
both containing T7lac promoter were used. These
plasmids contain a lac operator sequence just
downstream of T7 promoter. They also carry the
natural promoter and coding sequence for the lacI so
that the T7lac and lacI promoters diverge. When this
type of vector is used in DE3 lysogens to express the r-
fragment C, the lac repressor acts both at lacUV5
promoter in the host chromosome to repress the
transcription of the T7 RNA polymerase gene by the
host polymerase and at the T7lac promoter in the vector
to block the transcription of the target gene by T7 RNA
polymerase (Dubendorf and Studier, 1991; Studier,
1991). The results of the present study revealed that the
employment of BL21 (DE3) pLysS strain of E. coli as
an expression host and pET-28a and pET-22b as
expression vectors could overcome the probable toxic
effects of fragment C on the establishment of the
plasmid and the subsequent expression level of its
encoding gene. These results showed that the recently
optimized protocol set in our laboratory for high
expression level of ASNase II (Bahreini et al., 2014)
could be also applied for the expression of tetanus toxin
fragment C. Another important factor reducing the
overproduction of the recombinant proteins is the low
solubility rate (Gerday et al., 1990). It should be
mentioned that in some cases, the overexpression of a
special protein does not result in its overproduction.
The high-level expression of both heterologous and
host proteins in E. coli is often accompanied by the
formation of inclusion bodies, which are irreversible
aggregates mainly consisting of the overexpressed
polypeptide (Ho et al., 1990). Proteins in form of
inclusion bodies are insoluble agents resulting in low
production rate. In this regard, the protein expression at
low temperatures would be favorable to increase the
overproduction of the proteins, which tend to form
inclusion bodies. This is due to the fact that the
formation of inclusion bodies is sometimes avoided by
lowering the cultivation temperature (Vasina and
Baneyx, 1996). When E. coli is used as the host, the
cultivation temperature is sometimes decreased to
suppress the formation of inclusion bodies (Miyake et
al., 2007). A low-temperature expression system is also
expected to alleviate the heat denaturation of proteins
and would be suitable for the production of
thermolabile proteins (Miyake et al., 2007). Protein
expression at low temperatures is also useful for the
production of the enzymes whose activities are harmful
to the host cells, such as proteases that degrade the
essential components of the host, because the enzyme
activities can be suppressed by lowering the
temperature (Miyake et al., 2007). In the present study,
the transformants were induced with IPTG at four
different incubation temperatures (i.e., 37, 33, 30, and
25 °C) and three different incubation times (i.e., 1-3 h).
The optimal expression condition, in this study, was
acquired at 25 °C, 1 mM IPTG, and 3 h after IPTG
induction (Figure 4a), which was desirable to suppress
the formation of the inclusion bodies, resulting from the
overproduction of r-fragment C in E. coli expression
system. Ribas et al. (2000) similarly reported the
optimal expression condition at 1 mM IPTG and 3 h
Aghayipour & Teymourpour / Archives of Razi Institute, Vol. 73, No. 1 (2018) 27-38 36
after IPTG induction. In contrast with our results,
(Yousefi et al., 2013) reported the optimal expression
time of 8 h after IPTG induction. The transformed E.
coli BL21 (DE3) pLysS with pET-28a produced a
fusion protein (~54 kDa) consisting of a 6-histidin tag
and a thrombin cleavage site in N-terminal proportion,
and the cells transformed with pET-22b produced a
protein (~ 51.6 kDa) in a native form. The presence of a
histidin tag in the expressed protein facilitates its
purification via one-step metal-affinity chromatography.
On the other hand, as any additional amino acid
sequence may affect the structure, spatial conformation,
and the subsequent function of the native protein, they
should be removed from the native protein sequence in
order to be used in the clinical trial testing and final
product manufacturing. In this regard, the presence of
thrombin cleavage site (N-Leu-Val-Pro-Arg-Gly-Ser-
C) within the expressed protein produced in the present
study enabled us to cleave the 6-histidin tag from
protein sequence in the future studies. Although the
western blot analysis confirmed the expression of both
recombinant native and fusion fragments C, the yield
obtained by pET28a was higher than that of pET22b
(9.8% versus 8.5%, P<0.5). Therefore, it seems that
BL21 (DE3) pLysS, as an expression host, was more
compatible with pET28a expression vector than with
pET22b to express the r-fragment C. The results of the
present study revealed the occurrence of no mutations
or polymorphisms during gene amplification or cloning
procedure, which is a desirable characteristic for a
bacterial strain to be applied as a vaccinal strain.
Ethics
I hereby declare all ethical standards have been
respected in preparation of the submitted article.
Conflict of Interest
The authors declare that they have no conflict of interest.
Grant Support
This study was financially supported by Razi Vaccine
and Serum Research Institute, Karaj, Alborz, Iran.
Acknowledgement
We appreciate the staff of the Departments of
Genomics and Genetic Engineering, DTP, and
Anaerobic Vaccines, Razi Vaccine and Serum Research
Institute, Karaj, Alborz, Iran
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